Treatment of neurodegenerative conditions

ABSTRACT

A method is provided for treating a patient in need of therapy for a neurodegenerative disease comprising administering to that patient a therapeutically effective dose of a triglyceride oil containing both γ-linolenic acid and linoleic acid residues as triglyceride ester, the ratio of γ-linolenic acid to linoleic acid residues at the sn-2 position of the triglyceride being at least 0.8; the amount of γ-linolenic acid residues at the sn-2 position being at least 18%, wherein the oil is administered at a dose sufficient to maintain or elevate TGF-β1 levels in the patient at a therapeutic level. Preferably the method is that wherein the therapeutic level is such as to produce a TGF-β1/TNF-α ratio of at least 0.5 in blood of a patient, after 18 months of daily dosing. Preferred oils are Borage or Mucor oils having at least 35% of the sn-2 position fatty acid residues as γ-linolenic acid.

The present invention relates to a method for treating neurodegenerativeconditions, particularly those in which increase in transforming growthfactor β (TGF-β) is beneficial, particularly TGF-β1. More particularlythe present invention provides treatment for conditions such as multiplesclerosis and the degenerative sequelae associated with head trauma,stroke and intracranial bleeds, whereby neuronal function is improved orrestored from an impaired condition. Further provided are novel use ofknown and novel compounds comprising unsaturated fatty acid moieties forthe manufacture of medicaments capable of effectively treating suchconditions, more particularly being capable of achieving previouslyunattained levels of success with regard to recovery of neurologicalfunction.

It is well reported in the literature that essential fatty acids (EFAs)of the n-3 and n-6 unsaturation pattern have beneficial effect in a widevariety of human physiological disorders. WO 02/02105 (Laxdale Limited)describes their beneficial use for an extremely wide range of diseasesand as a general nutritional supplement. Harbige (1998) Proc. Nut. Soc.57, 555-562 reviewed the supplementation of diet with n-3 and n-6 acidsin autoimmune disease states, and particularly noted evidence of benefitof γ-linolenic (GLA) and/or linoleic acid (LA) rich oils, such as borageoil, in reducing clinically important signs and symptoms of rheumatoidarthritis.

Two studies on multiple sclerosis (MS) patients are noted that indicatethat relapse and severity of the disease might be reduced by treatmentwith oils containing n-6 acid moieties (Miller et al (1973) and Bates etal (1978)), but a further study failed to confirm this effect (Paty etal (1978). These papers report that supplementation of human patientswith about 20 g/day of linoleic acid (18:2 n-6) affected duration andseverity of relapses of multiple sclerosis such that relapses were lessfrequent, less severe and of shorter duration than controls. Bates notedthat a mixture of linoleic acid and γ-linolenic acid had been suggestedback in 1957 to be possibly more efficacious in treating inflammationand autoimmune diseases and set out to investigate this in the trial.However, it was found that where this combination was tried, at 3 g oilper day (Naudicelle Evening Primrose oil) patients who had relapsesbecame more ill on the trial oil than on the control.

Meta analysis of these linoleic acid studies by others (Dworkin et al(1984)) demonstrated reduced relapse rate and severity with a decreasein the degree of long-term progression of the disease in patients withmild multiple sclerosis. Later open studies of patients with multiplesclerosis suggest that low fat diet and/or manipulation of dietary n-3and n-6 fatty acids may be beneficial (Swank & Grimsgaard (1988);Harbige et al (1990).

Although the aetiology of MS remains unknown, strong evidence suggeststhe presence of autoimmune mechanisms in the disease pathogenesis[Martino & Hartung 1999]. Studies have shown that MS patients have amuch higher number of neuro-antigen e.g. myelin basic protein (MBP) andmyelin oligodendrocyte glycoprotein (MOG) autoreactive T-cells which arein an increased state of activation compared with healthy controls[Fredrikson et al 1994, Kerlero de Rosbo et al 1993, 1997, Chou et al1992, Ota et al 1990, Burns et al 1999, Zhang et al 1994, Tejada-Simonet al 2001]. The actual processes of axonal damage e.g. chronicinflammation, demyelination and astrogliosis in MS is complex but whitematter inflammation and demyelination are considered to determinedisease severity, whilst recent studies suggested that axonal damage inMS begins in the early stages of the disease and contributes todisability (De Stefano et al, 2001).

Experimental autoimmune encephalomyelitis (EAE) is the most frequentlyused animal model for immune mediated effects of MS. Studies in theguinea-pig have shown that linoleic acid partially suppresses theincidence and severity of EAE (Meade et al (1978)). Using γ-linolenicacid-rich oils from fungal or plant sources, complete protection wasdemonstrated in both rats and mice (Harbige et al (1995), 1997b). Theseinvestigations demonstrated disease modifying effects of linoleic acidand γ-linolenic acid on clinical and histopathological manifestations ofEAE. Depending on dose, γ-linolenic acid was fully protective in acuterat EAE whereas linoleic acid had dose-dependent action on the clinicalseverity but did not abolish it.

Despite these experimental findings, it is recognised that the humandisease, multiple sclerosis, is highly complex and can be converselyexacerbated and ameliorated by the activity of T-cells and other immuneresponse factors. It is thought that the n-6 fatty acids promoteautoimmune and inflammatory disease based upon results obtained withlinoleic acid only. TGF-β and PGE₂ production has been shown to beincreased non-specifically in γ-linolenic acid fed mice ex vivo; butwhilst TGF-β has been reported to protect in acute and relapsing EAE((Racke et al (1993); Santambrogio et al (1993)), PG inhibitors such asindomethacin augment, and thus worsen, the disease (Ovadia & Paterson(1982)).

Cytokines are implicated in the pathogenesis of MS, with many studiesshowing an increase in myelinotoxic inflammatory cytokines (TNF-α, IL-1βand IFN-γ) coinciding with the relapse phase of the disease. Conversely,levels of the anti-inflammatory and immunosuppressive cytokinetransforming growth factor-beta1 (TGF-β1) appear to be reduced during aphase of relapse and increase as the patient enters remission. Thus thebalance between biologically active TGF-β1 and the pro-inflammatoryTNF-α, IL-1β and IFN-γ appears to be dysregulated during MSrelapse-remission.

During natural recovery phase from EAE, TGF-β-secreting T-cells inhibitEAE effector cells, TGF-β is expressed in the CNS and, inoral-tolerance-induced protection in EAE, TGF-β and PGE₂ are expressedin the brain (Karpus & Swanborg (1991); Khoury et al (1992)). Harbige((1998) concluded that dietary γ-linolenic acid effects on EAE aremediated through Th₃-like mechanisms involving TGF-β and possiblythrough superoxide dismutase antioxidant activity.

It has been suggested to use, inter alia, γ-linolenic acid and linoleicacid rich Borage oil as a means to provide immuno-suppression inmultiple sclerosis (U.S. Pat. No. 4,058,594). The dose suggested is 2.4grams of oil per day and no actual evidence of efficacy is provided.

Borage oil (typically 23% γ-linolenic acid and 37% linoleic acid per100% fatty acid content) has been shown to significantly reduceclinically important signs and symptoms of autoimmune disease associatedwith active rheumatoid arthritis (Leventhal et al (1993)). Borage oiland fungal oil (see FIG. 1) have been shown to be effective in the EAEanimal model use to identify MS candidates, whilst never having beenshown to be significantly effective in the human disease. High levels oflinoleic rich oil containing low levels of γ-linolenic acid (EPO:linoleic acid:γ-linolenic acid 7:1) partially suppressed the incidenceand severity of EAE in rat (Merlin & Stackpoole, 1978) whereas theNaudicelle study referred to above led to worsening of patients. Inspite of the use of Borage oil and other GLA/LA containing oils such asEvening Primrose oil by multiple sclerosis sufferers over the past 30years or so, the vast majority of patients fail to recover from thedisease, showing no significant improvement, with the underlying diseasecontinuing to progress to death.

Other more dramatic immunosuppressant treatments, including T celldepleters and modulators such as cyclophosphamide, are also shown to beeffective in the EAE model, but where these are employed in the humanmultiple sclerosis disease symptoms improve, but the underlying diseasecontinues to progress. T-cells indeed produce beneficial cytokines, suchas TGF-β1, as well as deleterious ones in man. David Baker of Instituteof Neurology, UK summed up the disparity between what is effective inthe EAE and in MS with a paper entitled ‘Everything stops EAE, nothingstops MS’ at the 10 May 2004UK MS Frontiers meeting of the UK MSSociety.

It is clear that immunosuppression alone cannot cure MS. This is almostcertainly due to a fundamental underlying metabolic disorder in MSpatients that leads to membrane abnormality, cytokine dysregulation andsubsequent immune attack and lesioning. Although patients go intoremission in relapse-remitting disease, the underlying demyelinationproceeds.

The ‘gold standard’ treatment for MS remains interferon, such as withβ-Avonex®, Rebif® and other interferon preparations. This gold standardtreatment only addresses needs of some, eg 30%, of the patients and evenin these symptom improvement is restricted to reduced severity ofrelapses. Whilst symptoms may be reduced in a proportion of patients,the disease tends to progress to further disability and death due tounderlying degeneration.

The present inventors have now surprisingly determined that withcompliance to a ‘high dose’ treatment with triglyceride oil containingγ-linolenic acid with suitable accompanying fatty acid content,remarkable levels of improvement in almost all symptoms of MS can beachieved, way surpassing that provided by the present gold standardtreatment. Such success is particularly surprising in the light of theprior use of other γ-linolenic acid containing preparations without suchsignificant success.

Over an 18-month period, patients taking high dose selected borage oilshowed significant (p<0.001) and marked improvements in EDSS score, areduced rate of relapse, symptomatic relief of muscle spasticity andpainful sensory symptoms, and improved objective measures of cognitivefunctions. Low dose borage oil was without effect.

Patients taking high dose borage oil maintained their level ofperipheral blood mononuclear cell production (PBMC) of TGF-β1 during thetrial period, their pro-inflammatory cytokines TNF-α and IL-1β weresignificantly and markedly (<70%) reduced and they either maintained orincreased the PBMC membrane long chain omega-6 fatty acidsdihomo-γ-linolenic acid (DHLA) and arachidonic acid (AA) in contrast topatients taking placebo who demonstrated loss of these fatty acids overthe course of the trial period.

This whilst immuno-suppression would be expected to reduce activelesioning and neurodegeneration, the present treatment has apparentlytargeted maintenance of key membrane lipid components that are otherwisespecifically lost in MS, suggesting a correction of a metabolic defectnot otherwise effectively treated by current therapies. The fact thatthe low dose (5 grams/day) had no effect on this supports suchdetermination.

Particularly the inventors have determined that a triglyceride oilcomprising triglycerides of both γ-linolenic acid and linoleic acid withspecific positional distribution within the triglyceride molecules,preferably with oleic acid, can provide significant decreasing EDSSscore in multiple sclerosis patients over a number of months and years,a result that is unattainable with any of the currently administeredtherapies.

γ-Linolenic acid (18:3n-6, GLA) is known to be rapidly converted tolonger-chain omega-6 polyunsaturated fatty acids dihomo-γ-linolenic acidand arachidonic acid in vivo (Phylactos et al 1994, Harbige et al 1995,2000). Therefore to increase the level of membrane long chain omega-6fatty acids in MS the inventors have reviewed results obtained withseveral GLA-containing oils:—both fungal (from Mucor javanicus) andplant (Borago officianalis), Evening primrose Oenothera spp. orBlackcurrant Ribes spp) as well as a synthetic tri-GLA oil as GLAdelivery systems in an in vivo experimental animal model of MS known aschronic relapsing experimental autoimmune encephalomyelitis (CREAE).

Experimental autoimmune encephalomyelitis (EAE) is an autoimmuneinflammatory disease of the CNS, with or without demyelination,inducible in rodents and other mammalian species. Induction of EAE inrats however (using guinea pig basic protein), does not producehistological features of demyelination (Brosnan et al 1988) but inducesan acute mono-phasic disease pattern, unlike MS which is characterisedby CNS demyelination and is clinically relapsing-remitting. Chronicrelapsing and demyelinating EAE models (CREAE), which are characterisedby demyelination and relapse phases, are therefore currently the animalmodels of choice for MS research (Fazakerley 1997). With thedemonstration that myelin oligodendrocyte glycoprotein (MOG) is animportant neuroantigenic target in MS (Genain et al 1999) and thedemonstration of far greater responses of peripheral blood auto-reactivelymphocytes to this neuroantigen, compared with MBP, in MS (Kerlero deRosbo et al 1993, 1997) MOG induced CREAE has become the animal model ofchoice with features closely resembling those observed in MS(Fazalcerely et al 1997, Genain et al 1999, Amor et al 1994).

Based on the results of these experiments two key selection criteriawere adopted for selection improved lipid compounds for achieving thecurrent aims. Evidence from CREAE and rat EAE feeding studies indicatesthat an enriched blackcurrant seed oil (72% w/w 18:3n-6, GLA) did notprotect against EAE (see Table 3). Importantly blackcurrant seed oil hasa low sn-2 GLA with most of the GLA in the sn-1 and sn-3 positions(Lawson and Hughes 1988). Furthermore a structured triacylgcerolcontaining three GLA's (TG-GLA) provided protective effects similar tothat of the borage oil used in CREAE (Table 2). This would also beconsistent with the sn-2 GLA being important i.e. the outer pair sn-1and sn-3 GLA being enzymatically removed in vivo and probably undergooxidation leaving the sn-2 GLA only. This selective hydrolysis arisesfrom the known ability of specific lipases to remove the sn-1 and sn-3fatty acids from triacylgycerol molecules but an apparent protection ofthe sn-2 position in vivo (Lawson and Hughes 1988, Kyle 1990).

The inventors' review of this data also indicates that the ratio oflinoleic acid (LA) to γ-linolenic acid (GLA) residues may be a keyefficacy feature of oils containing LA and GLA in the CREAE model of MS(Table 1). Table I shows the compositional analysis and efficacy inCREAE of fungal oil, borage oil, evening primrose oil and safflower oil.The most effective treatment in reducing the incidence of CREAE wasfungal oil with an LA:GLA ratio of 0.85. Borage oil was also veryeffective with an LA:GLA ratio of 1.5. Furthermore, experiments with astructured triglyceride containing GLA at sn-1, sn-2 and sn-3 (TG-GLA)demonstrated GLA to be an active component. Moreover TG-GLA was alsoeffective at a lower dose level than the borage oil (see Table 2).

Different Borage seed oil's also appear to vary in their level of sn-2GLA e.g. 10% sn-2 GLA (Liu et al 2000) and 40% sn-2 (Lawson and Hughes1988) which is consistent with our unpublished observations of sn-2 GLA(range 38-46%) and possibly the failure of some borage oils to producefully protective effects in CREAE, although other factors such asantioxidant composition may also be important (unpublished). Borage oilshaving as much as 60% sn-2 GLA have been reported (Huang and Mills(1996) γ-Linolenic acid: metabolism and Its Roles in Nutrition andMediceine: Chapter 6) and noted to be effective at getting GLA intolymph.

Table 3 of EP 0520624 (Efamol Holdings) compares the triglyceridecontent of Evening Primrose and Borage Oils, the former being taught tobe more therapeutically effective than the latter for a variety of GLAresponsive disorders. This document indicates Borage oil to have twentyseven different triglyceride components, only 20% of which have sn-2GLA. Page 3, lines 40-42 notes that biological testing has shown thatequal amounts of GLA may indeed have very different effects when thatGLA is supplied as different oil sources. Crucially, it then directs thereader to one particular fraction present in Evening Primrose Oil (EPO),but not Borage Oil, as being responsible for the former's superioreffect in raising PGE1 (see EP 0520624 Chart page 4 and Table 2) andthus anti-inflammatory effect: that fraction beingdi-linoeoyl-mono-gamma-linolenyl-glycerol (DLMG) which it states to be18 to 19% of the total triglyceride in EPO. Page 6 clearly teaches thatthe position of the GLA, in sn-1, 2 or 3, is not important to thiseffect.

Dines et al (1994) Proceedings of the Physiological Society, AberdeenMeeting 14-16 Sep. 1994 report on studies treatment of diabeticneuropathy neuronal damage with γ-linolenic acid containing oils of thetype advocated by EP 0520624 and again note that Borage Oil was not veryeffective in treating this neurodegeneration whereas Evening primroseoil was. The paper concludes that Borage Oil contains other constituentsthat interfere with GLA activity.

In contrast to this prior art, the present inventors used a borage oilthat was selected with the highest sn-2 GLA for trial purposes(>40%)—compared with lower amounts in other samples that were availableat the time. Blackcurrant seed oil, which at the time was available inrelative large quantities was not considered optimal because of it's lowsn-2 GLA content.

A further selection criterion was that the level of total long chainmonoenoic fatty acids be kept below 5%. There was a significant level oferucic acid (22:1n-9) i.e. 1.4-2.38% of the total fatty acids, and otherlong chain monoenoic fatty acids i.e. 24:1n-9 (nervonic acid) and20:1n-9 (gadoleic acid) in different borage seed oil (Boragoofficinalis) samples from different sources (Table 4).

Additionally because of potential impact on absorption, metabolism andimmune functions of vitamin E (Harbige 1996, 2003), the trial oilcontained only natural levels of vitamin E (0.05 mg/g) with noadditionally added vitamin E as is routinely the case with commercialborage seed oils (e.g. 1 mg/g).

It is believed that such selected oils have an immunosuppressant effect,but significantly also have and a metabolic supplementation effect thathas benefit in reducing immune attack on lesions whilst creating theconditions necessary for their repair, something that has not beenachieved with any medication previously provided for MS.

In a first aspect of the present invention there is provided a method oftreating a patient in need of therapy for a neurodegenerative diseasecomprising administering to that patient a therapeutically effectivedose of an oil containing both γ-linolenic acid and linoleic acidresidues as triglyceride ester, the ratio of γ-linolenic acid tolinoleic acid residues at the sn-2 position of the triglyceride being atleast 0.8; the amount of γ-linolenic acid residues at the sn-2 positionbeing at least 18%, wherein the oil is administered at a dose sufficientto maintain or elevate TGF-13 levels in the patient to therapeuticlevels.

By therapeutic levels is meant levels at least consistent with healthysubjects. Preferably the dose is such as to produce a TGF-β1/TNF-α ratioin blood of a patient, after 18 months of daily dosing, of 0.4 to 3.0,at least 0.5, more preferably at least 0.75 and most preferably atleast 1. Preferably the dose is such as to produce a TGF-β1/IL-1β ratioin blood of a patient, after 18 months of daily dosing, of at least 0.5,more preferably at least 0.75 and most preferably at least 1. Preferablysaid levels are produced after 12 months and more preferably after 6months.

Typically the amount of oil administered daily will be between 3 and 30grams, orally dosed, still more preferably between 5 and 20 gams andmost preferably between 7 and 18 grams, typically 15 grams.

Most preferably, in addition to the γ-linolenic acid and linoleic acidfatty acid residues, the oil includes an esterified fatty acid that isnon-structural, ie. that is metabolised to produce energy, such as oleicacid residues. By residue is meant the moiety that remains after thefatty acid carboxyl group esterifies to one of the hydroxy groups of theglycerol molecule.

Most preferably the oil administered is an oil source from Borage oil ora fungal oil eg. eg from Mucor javanicus,

Typical Borage oil and fungal oil compositions are illustrated in Table1 wherein 18:2n-6 and 18:3n-6 represent linoleic and γ-linolenic acidresidue by percent respectively.

Typically Borage oils contain from 20 to 25% γ-linolenic acid residuesas percentage of fatty acid residues in the oil and from 35 to 40%linoleic acid residues. Preferred Borage oils are those in which theamount of esterified γ-linolenic acid at the sn-2 position is at least35% of fatty acid residues at that position, more preferably greaterthan 39% and still more preferably greater than 40%. Most preferred oilsare over 41%, such as 42 to 44% sn-2 GLA, whilst ideally they will beover 45%. As stated by Huang et al above, 60% sn-2 GLA Borage Oils havebeen produced and should be available for selection. The sn-1 and sn-3position residues are preferably linoleic, oleic and γ-linolenic acidresidues, with preferred oils having relatively high oleic acid contentin at least one, if not both, of these positions, eg, in excess of 12%,more preferably in excess of 14%.

A typical Borage oil suitable for use in the use of the presentinvention has fatty acid distribution as follows

Sn-1: 14% 18:1 (Oleic), 54% 18:2n-6 (linoleic) and 4% 18:3n-6(γ-linolenic)

Sn-2: 14% 18:1 (Oleic), 42% 18:2n-6 (linoleic) and 40% 18:3n-6(γ-linolenic)

Sn-3: 19% 18:1 (Oliec), 18% 18:2n-6 (linoleic) and 30% 18:3n-6(γ-linolenic)

Where a fungal oil is use, such as from Mucor species, the total amountof γ-linolenic acid residues may be lower than for Borage oil as long asthe sn-2 γ-linolenic acid:linoleic acid ratio is at least 0.8, morepreferably greater than 1. This is because fungal oils tend to have more‘metabolic’ directed oleic acid residues than linoleic acid residues.Thus preferred fungal oils are those in which the amount of esterifiedγ-linolenic acid at the sn-2 position is at least 18% of fatty acidresidues at that position, more preferably at least 20% and mostpreferably at least 22%. Preferred fungal oils have in excess of 45% ofthe sn-2 fatty acid residues as oleic acid residues, more preferably inexcess of 50%.

Sn-1: 25% 18:1 (Oleic), 5% 18:2n-6 (linoleic) and 13% 18:3n-6(γ-linolenic)

Sn-2: 54% 18:1 (Oleic), 19% 18:2n-6 (linoleic) and 20% 18:3n-6(γ-linolenic)

Sn-3: 40% 18:1 (Oliec), 3% 18:2n-6 (linoleic) and 20% 18:3n-6(γ-linolenic)

It will be realised by those skilled in the art that such oils will needto be sourced by testing for the percentage of said fatty acids at eachposition as an average over the many triglycerides in a give oil mix.Such is well within the skill of those in the art, such as eg.Mylnefiled Research Services Ltd, Lipid Analysis Unit, Mylnefiled,Inverghowrie, Dundee DD2, 5DA, Scotland UK. The applicants have managedto source a number of such oils meeting the criteria set out above, thehighest sn-2 figures of about 46% being found for example in New Zealandsourced oils in 2003: this of course may vary from year to year. It isimportant, however, given the lack of response with the low dose (5g/day) Borage Oil that a patients efforts to recover are not underminedby the equivalent of underdosing by provision of a lower sn-2% GLABorage Oil than the daily dose requires.

A further NMR method for analysis of such oils in a selection process isprovided in the methods section below. It will however be realised thatshould all oils available at a given time be below the 35% sn-2 GLAfigure, and preferably if they are below 40 or 45%, supplementation witha synthetic triglyceride or triglyceride mix will be possible. A numberof suitable lipids are known in the art and may for example be isolatedor combined mixtures of LGL, OGO, OGL, LGO or other components known tobe present in Borage Oil (see Table 3 of EP 0520624). Even TriGLA mightbe added (FR 2,617,161(1988)), although it is preferred for the presentpurposes to keep the sn-1 and sn-2 position GLA levels from getting toohigh as that would un the risk of overly pro-inflammatory effect due tooverflow into systemic GLA and thus DHGLA and then Arachidonic pools.Synthesis of OGO is for example taught in Y.-S. Huang, X. Lin, P. R.Redden and D. F. Horrobin, J. Am. Oil Chem. Soc., 72, 625-631 (1995) Invitro Hydrolysis of Natural and Synthetic γ-Linolenic Acid-ContainingTriacylglycerols by Pancreatic Lipase and K. Osada, K. Takahashi, M.Hatano and M. Hosokawa, Nippon Suisan Gakkaishi., 57, 119-125 (1991).Chem. Abs. 115:278299 Molecular Species of Enzymically-synthesizedPolyunsaturated Fatty acid-rich Triglycerides.

For treatment regimes where high amounts of any of these high Sn-2 GLAoils are administered it is recommended that the amount of potentiallytoxic long chain monoenoic fatty acids, such as erucic acid (22:1n-9)and other long chain monoenoic fatty acids i.e. 24:1n-9 (nervonic acid)and 20:1n-9 (gadoleic acid), are as low as possible, preferably lowerthan 5% of fatty acid residues, more preferably less than 3% and morepreferably less than 2%.

Another feature of a preferred oil is low or zero added vitamin E suchthat only natural levels of vitamin E (0.05 mg/g) is provided.

Further aspects of the present invention provide use of triglycerideoils as described above for the manufacture of a medicament for thetreatment of neurodegenerative disease, more specifically for thearresting of underlying neurodegeneration and the restoration ofneuronal function. Particularly such medicaments are for the normalisingof neuronal membrane composition, the restoration of healthy TGF-β1/TNFαratios and the ratios of TGF-β1 with other cytokines, the arresting ofneurodegeneration in multiple sclerosis and the restoration, in part orcompletely, of neuronal function such as measured, eg. By MRI or CATscan or by EDSS score. Such use will include treatment of cerebralimpairment after stroke, head trauma and intracranial bleeding.

Also provided are selected triglyceride oils having particular efficacyin treating multiple sclerosis and effecting beneficial changes in ratioof cytokines in vivo, these oils eing those set out as preferred for themethod described above. The oils for use in the present invention may beadministered by any of the conventional vehicles known in pharmacy. Mostconveniently they are administered as neat oils or in admixture withfoodstuffs, in the form of capsules containing such oils, or inenterically coated forms. Other forms will occur to those skilled in theart as delivery technology advances.

It will be realised by those skilled in the art that other beneficialagents may be combined with the oils for use in the present invention.These might be ion channel blockers, eg. sodium channel blockers,interferons, T-cell depleters, steroids or other palliative agents. Itwill further be realised that where the immune and inflammatoryresponses are being modulated, such combinations will need to be madecarefully, given the complex nature of these systems. However, given thedelayed response to the present oils, shorter acting agents might bebeneficial in the first months of treatment before the TGF-β1 levels arenormalised, as long as the additional treatment does not impede thisnormalization process.

The present invention will now be described by way of Example only byreference to the following non-limiting Tables, Examples and Figures.Further embodiments falling within the scope of the invention will occurto those skilled in the art in the light of these.

Tables

Table 1: Shows the compositional % Total fatty acid content of varioustriglyceride oils and protective effect in EAE.

Table 2: Shows the parameters of the three treatment groups in high sn-2GLA Borage Oil trial

Table 3: Shows the effect of various forms of GAL on EAE incidence andclinical score in SJL mice.

Table 4: Shows the failure of enriched Blackcurrent oil, a high GLA, butlow sn-2-GLA, plant oil, to match fungal and Borage oils in EAE.

Table 5: Shows the results analyses of four batches of trial Borage Oilparticularly with respect of monoenes.

Table 6: Shows an analysis of a non-trial oil particularly with respectto monoenes.

FIGURES

FIG. 1: Shows peripheral blood mononuclear cell cytokine production inplacebo and trail oil treated human MS patients at 18 months.

FIG. 2: Shows the effect of placebo and low dose (5 g/day) high sn-2 GLABorage oil on human MS patient EDSS score as compared to high dose (15g/day) displayed as a histogram.

FIG. 3: Shows the effect of placebo, low dose and high dose high sn-2GLA Borage oil on human MS patient EDSS displayed as a graph.

FIG. 4: Shows the effect of placebo, low dose and high dose high sn-2GLA Borage oil on human MS patient Mean Relapse rate (%) as a histogram

FIG. 5: Shows the effect of placebo, low dose and high dose high sn-2GLA Borage oil on human MS patient Mean Relapse rate (%) as a graph.

FIG. 6: Shows the effect of linoleic acid:γ-linolenic acid ratio of oilsas compared to their protective effect on mice CREAE.

METHODS Positional Analysis of Gamma-Linolenic Acid (GLA) in Borne OilSamples by Quantitative-¹³C-NMR

Analytical methodologies for the determination of fatty acid compositionand positional distribution in triacylglycerols generally requirehydrolysis of the triacylglycerols by enzymes or chemical processes andsubsequent analysis of the mono- and diacylglycerol components bychromatographic techniques. These methods are destructive and do notallow the recovery of the original samples. The hydrolysis procedureusually gives rise to some acyl migrations, resulting in substantialerrors of the positional distribution.

There are several properties of ¹³C nuclear magnetic resonance (NMR)that make its application to positional analysis useful. First, thechemical shift is sensitive to the molecular structure, thereby givingrise to a spectrum where each nucleus is identified by a peak at aspecific frequency. The resolution of the nuclei in each environment isdetermined by the linewidth and the chemical shift differences betweenadjacent peaks. Second, the area under the peak, arising from eachnucleus, is proportional to the number of nuclei in that environmentbecause all ¹³C exhibit the same absorption. Therefore, the chemicalshift and the integrated area of each peak can be used for bothqualitative and quantitative measurements of each nucleus. Thirdly, thepreparation of the sample for this application is simple. Finally, NMRis a non-destructive technique that allows the sample to be recoveredfor other purposes. ¹³C NMR methods are normally based on analysing thecluster of signals for the carbonyl carbons. Two clusters of signals arenormally observed corresponding to acids in the sn 1,3- and 2-positions.They are usually readily distinguished since the two environments giverise to a separation of about 0.4 ppm. Within each of these twoclusters, there must be separate signals for each acid or groups ofacids. This criterion is most easily met when the acids havecarbon-carbon double bonds (i.e. are unsaturated) close to the carboxylgroup e.g. n=4, 5 or 6. The carbonyl carbon signals from acids of thesame double bond group (e.g. EPA and AA) will not normally bedistinguished. Such methods seemed to be valuable for analysingGLA-containing triacylglycerols and this proved to be the case

REFERENCES

-   M. M. Bergana and T. W. Lee, J. Am. Oil Chem. Soc., 73, 551-556    (1996)-   G. Vlahov, Magn. Reson. Chem., 36, 359-362 (1998)

Experimental Materials/Sample-Preparation

Monoacidtriacylglycerols were purchased from Sigma Chemicals andNu-Chek-Prep Inc:

Tripalmitin (Tri-16:0) Tristearin (Tri-18:0) Triolein (Tri-18:1n-9)Trilinolein (Tri-18:2n-6) Trigammalinolenin (Tri-18:3n-6) Trieicosenoin(Tri-20:1n-9) Trierucin (Tri-22:1n-9) Trinervonin (Tri-24:1n-9)Approx 180 mg of lipid in 700 uL of deuteriochloroform was usedthroughout the study.

¹³C-NMR-data

The proton-decoupled ¹³C NMR data with supressed NOE were collected at21° C. in a 5-mm broadband probe on a Jeol 500 MHz spectrometeroperating at 125.728 MHz. Waltz decoupling was the chosen mode ofdecoupling and was gated on only during the 14.89s acquisition time. Therelaxation delay was set at 30 secs and the pulse angle was 90°.Thespectral window used was ca. 35 ppm (from 173.5 to 172.6 ppm) with a 170ppm offset. The spectra were internally referenced to CDCl₃ at 77.0 ppm.Typically, the approximate number of scans collected for adequatesignal-to-noise ranged from 300 to 1200 scans, depending on thecomplexity of the mixture. The total acquisition time for theexperiments ranged between 1-4 h (Borage oil 1272 scans/4 h). Datapoints 65,536.

Calculations

The GLA carbonyl signals at the 2- and 1,3-positions were well separatedfrom all over carbonyl peaks in the spectra of the triacylglycerols.This allowed the ratio of 2-GLA/1,3-GLA to be determined withreliability and precision in all cases. The method was validated byanalysing a test mixture containing 8 triacylglycerols, includingtrigammalinolenin, of known proportions similar to those present inborage oil. Gross compositions had previously been determined by GLC.Two methods of calculation were employed. The first was a self-containedNMR method calculating the 2-GLA percentage as follows:

$\frac{{{Integral}\mspace{14mu} {of}\mspace{14mu} {sn}} - {2\mspace{14mu} {peak}\mspace{14mu} {for}\mspace{14mu} {GLA} \times 3 \times 100}}{{{{Total}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {sn}} - 1},{2\mspace{14mu} {and}\mspace{14mu} 3\mspace{14mu} {integrals}}}$

The second used the ratio of 2-GLA/1,3 GLA integrals from the NMR andgross GLA composition as determined by GLC as follows:

$\frac{\begin{matrix}{{{Integral}\mspace{14mu} {of}\mspace{14mu} {sn}} - {2\mspace{14mu} {peak}\mspace{14mu} {for}\mspace{14mu} {GLA} \times}} \\{\% \mspace{14mu} {GLA}\mspace{14mu} {from}\mspace{14mu} {GC}\mspace{14mu} {analysis} \times 3}\end{matrix}}{{{{Total}\mspace{14mu} {integrals}\mspace{14mu} {for}\mspace{14mu} {GLA}\mspace{14mu} {at}\mspace{14mu} {sn}} - 1},{2\mspace{14mu} {and}\mspace{14mu} 3}}$

Results from both computations were in agreement. We regard thecomposite NMR-GLC method as more precise since it uses the parametersfrom each method that can be measured with most accuracy. GLA is a majorcomponent of borage oil and therefore the 2-/1,3-GLA ratio can bedetermined by NMR with precision as the results show. GLC is generallybetter at determining the composition of even minor fatty acids than NMRbut cannot give positional information.

Summary

Details of the experimental data are given in the tables and spectra. Itis summarised below. Capsules D and B are sample capsules of provided inthe clinical trial reported below.

1. 8-TG-Test-mixture sn-2 GLA found 22.2% actual 22.6% error  1.8% (n) =2 2. Capsule-D Gross GLA found 21.4% (n = 3) by GLC sn-2 GLA found 42.5%(n = 2) by NMR error +/−1% 3. Capsule-B Gross GLA found 21.1% (n = 3) byGLC sn-2 GLA found 40.8% (n = 2) by NMR error +/−1%In these borage oil samples the GLA content in the sn-2 position is41-42% i.e. almost double (L95×) that of the gross composition. Aprobable typical sample reported in the literature shows an enrichmentof 1.8 times gross.The NMR method can be used to give reliable analytical data for GLA atthe sn-2 position. Indeed, it is particularly suited to borage oilbecause of the non-interference of other fatty acids present.Interestingly results from the NMR method are reported to agree withthose from the older derivatisation-chromatographic ones. Our previousestimate (40%) derived from these older methods also agrees with the NMRresults.

Treatment Examples

Twenty-eight active relapsing-remitting (two relapses in the preceding18 months) multiple sclerosis patients (ages ranging from 18 to 65 yrs)were entered into a double-blind placebo controlled trial to investigatethe effects of encapsulated borage oil on clinical activity andlaboratory parameters over 18 months. This oil was of high Sn-2γ-linolenic (GLA) content (>40% GLA at Sn-2) with low monene (eg. erusicacid) content and had no added Vitamin E, a known immunomodulator.

Patients were recruited from neurology out-patient clinics at two innercity hospitals; hospital informed consent was obtained on first(baseline) visit. Exclusion criteria include any form of steroid orimmunosuppressive drug treatment, pregnancy, hyperlipidemia, regular useof aspirin or related drugs and vitamin or fatty acid supplementationwithin the previous three months.

Only patients meeting all the following criteria were included in thetrial: (a) able to provide informed consent prior to treatment, with thefull understanding that consent may be withdrawn at any time withoutprejudice; (b) male or female out-patients aged 18 to 60 yearsinclusive; (c) have confirmed diagnosis of clinically definite relapsingMS; (d) have had at least three documented clinical relapses in the pasttwo years; (e) have a baseline Expanded Disability Scoring Scale (EDSS)score of 0.0-5.5 inclusive, provided they have well documentedexacerbations; and (f) healthy, apart from the MS-related symptoms, asconfirmed by the medical history, physical examination and clinicalchemistry, urine and haematological tests.

Patients were randomly allocated by the Pharmacy Department to one ofthree groups each containing 12 patients:

One clinical group (n=12) to receive placebo (5 g of Polyethylene Glycol400)

Second clinical group (n=12) to receive low-dose (5 g) refined Borageofcinalis

Third clinical group (n=12) to receive high-dose (15 g) refined Boragoofficinalis

Supplementation was in the form of one gram oil capsules daily (5/dayfor low dose, 15/day high dose) for 18 months duration. Boragoofficinalis oil and omega-6 polyunsaturated fatty acids are foodingredients that are generally recognised as safe for human consumption(GRAS). There are no classification or labelling requirements under ECregulations. Clinical assessment included: Extended Disability ScaleScores (EDSS) and clinical relapse record. Venous blood (50 mls) wasobtained for laboratory studies on the 1^(st), 3^(rd), 6^(th), 12^(th),15^(th) and 18^(th) month of supplementation.

The following biochemical and immunological parameters were investigatedon each visit for comparison with pre-treatment data and between groupdata:

-   -   Stimulated and unstimulated ex vivo peripheral blood mononuclear        cell cytokine production: TGF-β1, IFN-γ, TNF-α, IL-1β, IL-6 and        IFN-β, which are implicated in the pathogenesis of MS. Cytokine        and related gene expression.    -   Soluble adhesion molecules in serum particularly ICAM-1 and        VCAM-1    -   Peripheral blood mononuclear cell membrane fatty acids and        plasma phospholipid fatty acid composition.        Results are shown in Tables 1 and 2 and FIGS. 1 to 5.        The primary outcome parameter was the number of clinical        relapses between baseline (Month 0) and the end of treatment        (Month 18). Secondary outcome parameters included: the time to        first clinical relapse; severity of relapses, as assessed by        EDSS score and the use of steroid treatment; and changes in EDSS        at Month 3, 6, 9, 12, and 18 compared to baseline and defined as        at least 1.0 point increase in the EDSS that is sustained for 3        months or at least 1.5 point increase on the EDSS from the        baseline EDSS that is sustained for 3 months. As this trial did        not receive external funding, it was not possible for financial        reasons to evaluate MS diseases activity with magnetic resonance        imaging. 1 of 3

Eleven patients were in the placebo group, seven patients had beentaking low-dose Borage oil, and ten patients had been taking high-doseBorage oil. The study drug was well-tolerated, and there were no seriousadverse events during the 18-month trial.

Results

Two patients had developed diarrhea, both of whom were later confirmedto have been taking high-dose Borage oil. The diarrhea was mild in onepatient, but was moderately severe in the second patient, who laterdiscontinued the study drug. The code was not broken and the diarrheahad stopped after the discontinuation of the drug, but reappeared uponre-challenge. Therefore, this patient was withdrawn from the trial. Theremaining patients who were treated with high-dose Borage oil showedexcellent clinical improvement on all primary and secondary outcomecriteria. For example, their mean EDSS score after 6 months of treatmenthad improved from baseline EDSS (FIG. 1). More importantly, the meannumber of clinical relapses had significantly reduced after 6 months oftreatment when compared to the number of relapses in the placebo group(FIG. 2). In contrast, patients who had been receiving low-dose Borageoil did not show any clinical improvement when compared to the placebogroup. In addition to its beneficial effect on MS disease activity, highrinse Borage oil provided some symptomatic relief of muscle spasticity(stiffness) and painful sensory symptoms, and also improved cognitivefunctions.

As can be seen for the figures below, relapse rate after 9, 12 and 18months was down to zero in the high dose group. The increase seen at 15months was due to a patient dropping out of this group.

The following are three brief case histories to illustrate thetherapeutic benefits of high dose high sn-2 GLA Borage oil. The firsttwo are from the trial while the third is a post trial patient for whomMRI studies were obtained.

Patient 1 (Treatment):

The first patient was a 48 year old woman who had had a clinicallyactive, relapsing remitting MS for 9 years. She had originally worked asa full-time administrator at the local Health Authority, but she wasunable to perform her duties because of her severe MS. Therefore, shelater worked as a part-time secretary, but still had difficulties inmobilization because of muscles stiffness and sensory disturbances. Shewas also experiencing severe clinical relapses at an average of onerelapse every nine months. Most of these relapses had resulted inhospital admissions for steroid therapy. In view of her active MS, shewas recruited into the Borage oil trial. There were no adverse eventsrelating to the study, and after taking the medication for four months,she experienced good improvement in her walking and sensory symptoms.

About nine months after therapy, she was well enough to start full-timeemployment. In addition, she remained relapse-free for the 18-monthduration of the clinical trial. Following the conclusion of the trial,the treatment code revealed that she was taking high-dose Borage oil.

Patient 2 (Control):

The second case was a 46-year old woman who also had a clinically activerelapsing remitting MS for 8 years. She had originally worked as a shopassistant, but became unemployed after MS was diagnosed.

Her symptoms included difficulty with mobilisation and painful sensorysymptoms in both legs. She had experienced three clinical relapses inthe two years preceding the clinical trial, and had been admitted tohospital twice for steroid therapy. Consequently, she was recruited intothe Borage oil trial, but her walking continued to deteriorate. Sixmonths into the trial, she need to use a walking stick and also receivedtreatment with Baclofen to reduce low limb spasticity. Approximately tenmonths after starting the Borage oil trial, she was admitted to hospitalbecause of severe clinical relapse, which was treated with steroids. Shelater developed bladder disturbances and began to use a wheelchair forlong journeys. The treatment code was broken after the conclusion of the18-month trial, and she was found to have been taking placebo. Sincethen, she started using a walking frame for journeys exceeding 50 yards.

Patient 3: Treatment (additional to trial)

The third case was a 26 year-old man who was diagnosed with definite MSin April 2001. His symptoms had started in 1999 when he complained ofdiffuse, intractable pain affecting various parts of his body,particularly the left side of the chest and abdomen. This was followedby intermittent numbness in the hands and feet, associated withfluctuating weakness. There were also distressing bladder symptoms inthe form of urinary frequency and urgency. The diagnosis of MS in 2001was based on his relapsing remitting symptoms, and was confirmed bypositive cerebrospinal fluid analysis and magnetic resonance imaging(MRI) of the brain, which showed multiple white matter abnormalities inboth cerebral hemispheres. Symptoms did not respond to variouspharmaceutical therapies.

In April 2003, oral supplementation with the present high dose Borageoil was commenced. The patient reported dramatic improvement in hissymptoms within three months of starting this oral supplementation. Hispainful sensory symptoms disappeared completely. He reported no numbnessor weakness since May 2003, and noticed significant improvement in hisbladder control. The oral supplementation caused no adverse events. Arepeat brain MRI was undertaken to verify the reported improvement in MrN′s symptoms. The repeat MRI showed a reduction in the size anddistribution of the white matter abnormalities.

TABLE 1 Compositional (% Total FAs) Characteristics of Various Oils andtheir Protective Effects in EAE 18:2n-6/ INCIDENCE Treatment 18:2n-618:3n-6 18:3n-6 18:1n-9 OF EAE FGO 17 20 0.6 35 0/10 BOO 37 24 1.5 153/10 EPO 71 9.4 7.5 9 7/10 SAF 66 — — 17 9/10 Controls — — — — 9/10 FGO,Fungal Oil; BOO, Borage Oil; EPO, Evening Primrose Oil, SAF, SafflowerOil.

TABLE 2 Treatment Groups - Borage oil-MS trial Mean Relapse Rate Mean(in past Base Female Male two years) EDSS Number Group Placebo 7 4 2.63.9 11 Low Dose 5 2 2.9 3.5 7 High Dose 8 2 3.4 2.8 10 Total 20 8 2.93.4 28

TABLE 3 Molecular Species Comparison of Triacylglycerol-GLA (TG- GLA),Ethyl-Ester-GLA (EE-GLA) and Borago Officinalis Oil-GLA (BOR-GLA) inMOG-induced CREAE in SJL Mice Treatment No. with EAE Mean Clinical ScoreControl 10/11 3.3 ± 1.3  EE-GLA^(a) 5/6 3.0 ± 0.8  TG-GLA^(a) 3/6 1.0 ±1.3^(c) BOR-GLA^(b) 3/6 1.0 ± 1.2^(c) ^(a)Animals given 100 μl of testlipid; ^(b)250 μl BOR-GLA given. Significance of difference comparedwith controls, ^(c)p < 0.05

TABLE 4 Effect of enriched black-currant seed oil (73% GLA) on theincidence of EAE % Incidence of EAE (Days after immunisation) 13 17 21Controls (n = 10) 60 90 10 Blackcurrant (n = 10) 10 80 70 Note:Blackcurrant oil delays the incidence but does not provide fullprotection. Animals were fed 7 days after sensitization (immunisation).

TABLE 5 ANALYSIS REPORTS ON TRIAL BORAGE OIL (% Total Fatty Acids) FattyAcids % SAMPLE 1 SAMPLE 2 SAMPLE 3 SAMPLE 4 16:0 13.29 13.47 12.86 13.1116:1n7 0.21 0.22 0.21 0.21 18:0 3.50 3.47 3.54 3.50 18:1n9 16.22 16.2216.33 16.22 18:1n7 0.64 0.66 0.65 0.64 18:2n6 38.00 38.01 38.25 37.9618:3n6 22.59 22.66 22.69 22.56 18:3n3 0.18 0.18 0.17 0.19 20:0 0.20 0.180.20 0.21 20:1n9 2.96 2.88 3.06 3.06 22:1n9 1.55 1.41 1.50 1.58 24:1n90.60 0.63 0.52 0.71

TABLE 6 ANALYSIS OF A NON-TRIAL BORAGE OIL (% Total Fatty Acids) FattyAcids % Total Fatty Acids 16:0 11.07 16:1n-7 0.17 18:0 3.70 18:1n-916.37 18:1n-7 0.66 18:2n-6 37.71 18:3n-6 21.89 18:3n-3 0.17 20:0 0.2520:1n-9 3.79 ↑ 22:1n-9 2.38 ↑ (high) 24:1n-9 1.47 ↑

1. A method of treating a patient in need of therapy for aneurodegenerative disease comprising administering to that patient atherapeutically effective dose of a triglyceride oil containing bothγ-linolenic acid and linoleic acid residues as triglyceride ester, theratio of γ-linolenic acid to linoleic acid residues at the sn-2 positionof the triglyceride being at least 0.8; the amount of γ-linolenic acidresidues at the sn-2 position being at least 18%, wherein the oil isadministered at a dose sufficient to maintain or elevate TGF-β1 levelsin the patient at a therapeutic level.
 2. A method as claimed in claim 1wherein the therapeutic level is such as to produce a TGF-β1/TNF-α ratioof at least 0.5 in blood of a patient, after 18 months of daily dosing.3. A method as claimed in claim 2 wherein the ratio is at least 0.75. 4.A method as claimed in claim 2 wherein the ratio is at least
 1. 5. Amethod as claimed in claim 1 wherein the amount of oil administered isbetween 3 and 30 grams per day.
 6. A method as claimed in claim 1wherein the oil is administered orally.
 7. A method as claimed in claim1 wherein the dose is sufficient to administer at least 1 gram ofγ-linolenic acid residues, as residues in the sn-2 position, excludingother γ-linolenic acid content of the oil.
 8. A method as claimed inclaim 1 wherein the amount of γ-linolenic acid in the sn-2 position inthe dose of oil is sufficient to administer at least 2 grams of saidsn-2 γ-linolenic acid.
 9. A method as claimed in claim 1 wherein thedose is between 8 and 20 grams.
 10. A method as claimed in claim 1wherein in addition to the γ-linolenic acid and linolenic acid fattyacid residues, the triglyceride includes an esterified fatty acid thatis non-structural.
 11. A method as claimed in claim 10 wherein thetriglyceride contains oleic acid residues.
 12. A method as claimed inclaim 1 wherein the oil is that obtained from a fungus or a plantselected from the group consisting of Mucor and Borago species.
 13. Amethod as claimed in claim 1 wherein the fungus or plant is selectedfrom Mucor javanicus and Borago officianalis.
 14. A method as claimed inclaim 1 wherein the oil is a Borne oil in which the percentage ofesterified γ-linolenic acid at the sn-2 position is at least 35% offatty acid residues at that position.
 15. A method as claimed in claim14 wherein the percentage of esterified γ-linolenic acid at the sn-2position is at least 39% of fatty acid residues at that position.
 16. Amethod as claimed in claim 14 wherein the percentage of esterifiedγ-linolenic acid at the sn-2 position is at least 45% of fatty acidresidues at that position.
 17. A method as claimed in claim 1 whereinthe fatty acid residues in the sn-1 and sn-3 position include linoleic,oleic and γ-linolenic acid residues.
 18. A method as claimed in claim 1wherein the triglyceride oil has an oleic acid content in one or both ofthe sn-1 and sn-3 positions of in excess of 12%.
 19. A method as claimedin claim 1 wherein the oil is Mucor oil and, the total percentage ofesterified γ-linolenic acid residues at the sn-2 position is at least20% of fatty acid residues at that position.
 20. A method as claimed inclaim 19 wherein the triglyceride oil has in excess of 45% of the sn-2fatty acid residues as oleic acid residues.
 21. A method as claimed inclaim 19 wherein the triglyceride oil has in excess of 50% of the sn-2fatty acids as oleic acid residues.
 22. A method as claimed in claim 1wherein the triglyceride oil contains less than 5% monoenoic fatty acidresidues as % total fatty acid residues.
 23. A method as claimed inclaim 22 wherein the triglyceride oil contains less than 5% in totalerucic acid (22:1n-9), 24:1n-9 (nervonic acid) and 20:1n-9 (gadoleicacid) as a percentage of total fatty acid residues.
 24. A method asclaimed in claim 22 wherein the amount of said acid is between 1° A) and5% of fatty acid residues in the oil.
 25. A method as claimed in claim 1wherein the oil has no added vitamin E.
 26. A method as claimed in claim1 wherein the amount of Vitamin E is between 0 and 0.1 mg/g.
 27. Amethod as claimed in claim 1 wherein the neurodegenerative disease isarrested or neuronal function is restored.
 28. A method as claimed inclaim 1 wherein treatment is for multiple sclerosis or the degenerativesequelae associated with head trauma, stroke and intracranial bleeds.29. A method as claimed in claim 28 wherein the treatment repairslesions.
 30. A method as claimed in claim 1 wherein the treatment uses adose sufficient to relieve muscle spasticity and/or pain.
 31. A methodas claimed in claim 1 wherein the dosage is sufficient to improvecognitive function.
 32. A method as claimed in claim 1 wherein thedosage is sufficient to eliminate relapses.
 33. A method as claimed inclaim 1 wherein the dosage is sufficient to improve the patients EDSSscore by at least 1 unit over a period of 1 years treatment.
 34. Amethod as claimed in claim 1 wherein the dosage is sufficient to restoreEDSS of a patient with EDSS above 2.5 to below 2 over a period of 1years treatment.
 35. Use of an oil as described in claim 1 for themanufacture of a medicament for the treatment of neurodegenerative
 36. Apharmaceutical composition for the treatment of neurodegenerativedisease comprising a Borago or Mucor species triglyceride oil asdescribed in claim 14.